DNA sequences for ammonium transporter, plasmids, bacteria, yeasts, plant cells and plants containing the transporter

ABSTRACT

There are described DNA sequences, that contain the coding region of ammonium transporters, which after introduction in suitable vectors are introduced into the plant genome and lead to the formation of mRNA which makes possible either the formation of new ammonium transporters, or prevents the formation of endogenous ammonium transporters in transgenic plants. There are further described plasmids, bacteria, yeast strains, plant cells and transgenic plants, which contain the sequences of ammonium transporters as a constituent of a recombinant DNA, and also a process for the identification and isolation of DNA sequences, coding for ammonium transporters.

[0001] The present invention relates to DNA sequences that contain thecoding region of ammonium transporters, whose introduction into a plantgenome modifies the uptake and transfer of nitrogen compounds intransgenic plants, as well as plasmids, bacteria, yeasts, plant cellsand plants containing these DNA sequences, and also a process for theidentification and isolation of DNA sequences that code for the ammoniumtransporter.

[0002] The supplying of growing plants with nitrogen compounds is alimiting factor in biomass production and is thus a limit on the yieldof agricultural production. For this reason, nitrogen compounds, oftenin the form of mineral fertilisers, are added in agricultural biomassproduction.

[0003] An only partial uptake of the added nitrogen compounds by theplants makes it on the one hand necessary that the nitrogen fertiliserproduced with high energy input is used in an excess, and on the otherhand it leads only to a partial uptake so that the nitrogen compoundsare washed into the ground water which can lead to considerableecological problems.

[0004] There is thus a great interest in plants which are capable oftaking up large amounts of nitrogen as well as in the provision of thepossibility of modifying the nitrogen uptake in plants.

[0005] For many plants there is provided information that the uptake ofnitrogen is essentially in the form of nitrate salts. In strongly acidsoils or in soils which follow intensive cultivation or have a strongtannin content, the nitrate formation (nitrification) is howeverstrongly reduced and the uptake of nitrogen in the form of ammoniumbecomes the most important mechanism for the uptake of nitrogencompounds (Raven & Smith 1976 New Phytol 76: 415-431). Plants which arewell adapted to acid soils, appear in part to favour ammonium ratherthan nitrate uptake and can tolerate ammonium ion concentrations thatwould be toxic for other plants. Examples of these plants are sugarcane, Betula verucosa or Lolium rigidum (Foy et al 1978, Ann Rev PlantPhysiol 29: 511-566). The toxicity of the ammonium follows from adisplacement of the ion balance: the uptake of the positively chargedammonium ion leads to an acidification of the cytoplasm, provided thatno cations are secreted in counter-exchange. In an uptake of ammoniumvia a transport system whose uptake mechanism is based on an ammoniumion proton antiport, the ionic imbalance is not a problem.

[0006] There is thus a great interest in transport systems which work bythe mechanism of an ammonium ion proton antiport and/or in systems whichcould be converted through techniques of protein engineering intoammonium ion proton antiports.

[0007] In spite of extensive efforts, it has not been possible up untilnow to isolate transport systems with whose help plants are protectedagainst an ammonium ion loss caused by membrane diffusion (retrievalsystem).

[0008] By the term ammonium is also to be understood methylamine whichis analogous to ammonium.

[0009] An active uptake system has been investigated in the fungusAspergillus nidulans (e.g. Arst et al., 1973, Mol Gen Genet 121:239-245), as well as in Penicillum chrysogenum (Hackette et al., J BiolChem 245: 4241-4250). In these studies methylamine was used as theammonium analogue. For Aspergillus nidulans, five genetic loci wereestablished, which take part in the transport of methylamine (Pateman etal., 1973, J Bacteriol 114: 943-950). In biochemical experimentsconcerning the methylammonium transport in Penicillum chrysogenum andSaccharomyces cerevisisae, it has been shown that the transport istemperature and pH dependent, and that the pH optimum is 6.0 to 6.5 andthe transport efficiency steadily rises up to a temperature of 35° C.(Roon et al., 1975, J Bacteriol 122: 502-509). The methylamine and/orammonium transport in Saccharomyces cerevisiae is dependent on thesupply of easily usable chemical energy, e.g. in the of glucose. Thetransport system consists of at least three independent transporters,which differ in transport capacity and affinity for the substrate:besides a high affinity transporter with low capacity (Km value=250 μM,maximum speed Vmax=20 nmol/min per mg cells (dry weight)) there is a lowaffinity system with high capacity (Km=2 mM, Vmax=50 nmol/min per mgcells) and a low affinity system with medium capacity (Km=20 mM, Vmax=33nmol/min per mg cells) (Dubois & Grenson, 1979, Mol Gen Genet 175:67-76). In the presence of glutamine or asparagine in the surroundingmedium, the ammonium uptake is reduced by 60 to 70%, whilst other amineshardly have any influence (Roon et al., 1975, J Bacteriol 122: 502-509).

[0010] Nothing is known about the molecular nature of the ammoniumtransporter of the above-mentioned or different fungi and otherorganisms, such as for example bacteria. Equally nothing is known aboutsystems, with whose help fungi or bacteria protect against ammonium ionloss by membrane diffusion (retrieval systems) at the molecular level.Further genes, which code for ammonium transporters are not known.

[0011] Various evidence suggests that the ammonium transport inSaccharomyces cerevisiae is accomplished by at least two functionallydifferent transport systems (Dubois & Grenson, 1979, Mol Gen Genet 175:67-76):

[0012] 1. Kinetic analyses of the methylamine-uptake by Saccharomycesshow an abrupt transition between apparent linear sections, whereby bothfunctions are inhibitable by ammonium.

[0013] 2. Both functions can be separately excluded by mutation. Theresulting mutations mep-1 and mep-2 are genetically independent.

[0014] 3. The mutants mep-1 and mep-2 can each grow in media withammonium as the only nitrogen source, whilst a double mutant mep-1/mep-2shows hardly any growth under these conditions (data taken from Dubois &Grenson, 1979).

[0015] A clarification of the ammonium transport processes in plants,that leads to similar detailed information such as for yeast, is notavailable and because of the difficulty of the molecular biologicalanalysis of mutations is scarcely possible in a corresponding manner.

[0016] The object of the present invention is to provide, coding forDNA-sequences of ammonium transporters, which cause a change in theuptake and transfer of nitrogen compounds in transgenic plants.

[0017] The object of the present invention is further to provide,DNA-constructs, such as plasmids, with which the ammonium transport intransgenic plants can be modified by introduction of the correspondingconstruct (plasmids) into the plant genome which leads upontranscription to the formation of a new ammonium transporter molecule inthe transgenic plant and/or the suppression of the formation the plant'sown ammonium transporter molecules.

[0018] There have now surprisingly been found DNA sequences, thatcontain the coding region of a plant ammonium transporter, whereby theinformation contained in the nucleotide sequence when integrated in aplant genome

[0019] a) makes possible under control of a promoter in senseorientation, the expression of a translatable mRNA, which leads to thesynthesis of an ammonium transporter in transgenic plants, or

[0020] b) makes possible under control of a promoter in anti-senseorientation, the expression of a non-translatable mRNA, which preventsthe synthesis of an endogenous ammonium transporter in transgenicplants.

[0021] A further aspect of the invention are therefore DNA sequences,which contain the coding region of a plant ammonium transporter.

[0022] In an analogous way the ammonium transporter can also be used tomodify animal cells.

[0023] The identification of DNA sequences, which code for a plantammonium transporter can take place by a process, in which DNA sequencesare identified and isolated, which are able to complement specificmutations in the yeast Saccharomyces cerevisiae, especially mutations,which have the result that the corresponding strains cannot grow anyfurther in media which contain ammonium as the only nitrogen source.Such a strain can be transformed with a plant cDNA-library andtransformands can be selected which can grow in media which containammonium as the only nitrogen source.

[0024] A further aspect of the invention is a process for theidentification and isolation of DNA sequences from plants that code forammonium transporters, which includes the following steps:

[0025] a) transformation of a yeast strain, which cannot grow in mediawhich contain ammonium as the only nitrogen source, with a plantcDNA-library using suitable expression vectors,

[0026] b) selection and propagation of transformands, which afterexpression of plant cDNA-sequences, can grow in media which containammonium as the only nitrogen source, and

[0027] c) isolation of the expression vectors, which carry a plant cDNAinsert from the selected transformand.

[0028] The yeast strain in process step a) is preferably one whichcannot take up ammonium from the medium because of mutations in thetransport systems for the ammonium uptake. Preferred is the doublemutant mep1/mep2 (strain 26972c), described by Dubois & Grenson (1979,Mol. Gen. Genet. 175:67-76), and with which two uptake systems forammonium are interrupted following mutation.

[0029] A further aspect of the invention are DNA sequences from plants,which are obtainable using the above described process and which codefor a protein with the biological activity of an ammonium transporter.

[0030] Whether it is possible using such complementation process toidentify DNA sequences which code for plant ammonium transportersdepends on various factors. First the expression plasmid suitable foruse in yeast must contain cDNA fragments which code for the plantammonium transporter, that means the mRNA fundamental for the cDNAsynthesis must result from tissues which express the ammoniumtransporter. Since nothing is known about plant ammonium transporters,tissues which code for the ammonium transporter are therefore also notknow.

[0031] A further prerequisite for the success of the complementationstrategy is that ammonium transport systems existing in plants can befunctionally expressed in yeast, since only in this case is acomplementation of the deficiency liberated by the mutation possible.Since nothing is known about plant ammonium transporters, it is also notknown whether a functional expression in yeast is possible.

[0032] It has now further been surprisingly found that by expression ofa cDNA library, for example from leaf tissue of Arabidopsis thaliana, bymeans of expression plasmids suitable for use in yeast which contain thepromoter of phosphoglycerate kinase from yeast, the complementation ofthe double mutation mep-1/mep-2 is possible, if the expression plasmidscontain specified plant cDNA fragments. These cDNA fragments code forplant ammonium transporters.

[0033] The identification of plant ammonium transporters is describedhere using Arabidopsis thaliana as an example, but it is not howeverlimited to this plant species.

[0034] A cDNA fragment that codes for a plant ammonium transportercontaining for example the following sequence. (Seq. ID No.1):                            TTCTTCTCTAAACTCTCAAC 20 ATG TCT TGC TCG GCCACC GAT CTC GCC GTC CTG TTG CGT CCT AAT. 65 Met Ser Cys Ser Ala Thr AspLeu Ala Val Leu Leu Gly Pro Asn GCC ACG GCG GCG GCC AAC TAC ATA TGT GGCCAG CTA GGC GAC GTC 110 Ala Thr Ala Ala Ala Asn Tyr Ile Cys Gly Gln LeuGly Asp Val AAC AAC AAA TTC ATC GAC ACC GCT TTC GCT ATA GAC AAC ACT TAC155 Asn Asn Lys Phe Ile Asp Thr Ala Phe Ala Ile Asp Asn Thr Tyr CTC CTCTTC TCC GCC TAC CTT GTC TTC TCT ATG CAG CTT GGC TTC 200 Leu Leu Phe SerAla Tyr Leu Val Phe Ser Met Gln Leu Gly Phe GCT ATG CTC TGT GCC CGT TCCGTG AGA GCC AAG AAT ACT ATG AAC 245 Ala Met Leu Cys Ala Gly Ser Val ArgAla Lys Asn Thr Met Asn ATC ATG CTT ACC AAC GTC CTT GAC GCT GCA GCC GGTGGT CTC TTC 290 Ile Met Leu Thr Asn Val Leu Asp Ala Ala Ala Gly Gly LeuPhe TAT TAT CTG TTT GGC TAC GCC TTT GCC TTT GGA TCT CCG TCC AAT 335 TyrTyr Leu Phe Gly Tyr Ala Phe Ala Phe Gly Ser Pro Ser Asn GGT TTC ATC GGTAAA CAC TAC TTT GGT CTC AAA GAC ATC CCC ACG 380 Gly Phe Ile Gly Lys HisTyr Phe Gly Leu Lys Asp Ile Pro Thr GCC TCT GCT GAC TAC TCC AAC TTT CTCTAC CAA TGG GCC TTT GCA 425 Ala Ser Ala Asp Tyr Ser Asn Phe Leu Tyr GlnTrp Ala Phe Ala ATC GCT GCG GCT GGA ATC ACA ACT GGC TCC ATC GCT GAA CGGACA 470 Ile Ala Ala Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr CAGTTC GTG GCT TAC CTA ATC TAT TCC TCT TTC TTA ACC GGG TTT 515 Gln Phe ValAla Tyr Leu Ile Tyr Ser Ser Phe Leu Thr Gly Phe GTT TAC CCG GTC GTC TCTCAC TCG TTC TGG TCA GTT CAT GGA TGG 560 Val Tyr Pro Val Val Ser His TrpPhe Trp Ser Val Asp Gly Trp GCC AGC CCG TTC CGT ACC GAT GGA CAT TTG CTTTTC AGC ACC GGA 605 Ala Ser Pro Phe Arg Thr Asp Gly Asp Leu Leu Phe SerThr Gly GCG ATA GAT TTC GCT GGG TCC GGT GTT GTT CAT ATG GTC GGA GGT 650Ala Ile Asp Phe Ala Gly Ser Gly Val Val His Met Val Gly Gly ATC GCT GGACTC TGG GGT GCG CTC ATC GAA GGT CCA CGA CTT GGC 695 Ile Ala Gly Leu TrpGly Ala Leu Ile Glu Gly Pro Arg Leu Gly CGG TTC GAT AAC GGA GGC CGT GCCATC GCT CTT CGT GGC CAC TCG 740 Arg Phe Asp Asn Gly Gly Arg Ala Ile AlaLeu Arg Gly His Ser GCG TCA CTT GTT GTC CTT GGA ACA TTC CTC CTC TGG TTTGGA TGG 785 Ala Ser Leu Val Val Leu Gly Thr Phe Leu Leu Trp Phe Gly TrpTAC GGA TTT AAC CCC GGT TCC TTC AAC AAG ATC CTA GTC ACG TAC 830 Tyr GlyPhe Asn Pro Gly Ser Phe Asn Lys Ile Leu Val Thr Tyr GAG ACA GGC ACA TACAAC GGC CAG TGG AGC GCG GTC GGA CGG ACA 875 Glu Thr Gly Thr Tyr Asn GlyGln Trp Ser Ala Val Gly Arg Thr GCT GTC ACA ACA ACG TTA GCT GGC TGC ACCGCG GCG CTG ACA ACC 920 Ala Val Thr Thr Thr Leu Ala Gly Cys Thr Ala AlaLeu Thr Thr CTA TTT CCC AAA CGT CTA CTC TCG GGA CAT TGG AAC GTC ACT GAT965 Leu Phe Gly Lys Arg Leu Leu Ser Gly His Trp Asn Val Thr Asp GTA TGCAAC GGC CTC CTC GGA GGG TTT GCA GCC ATA ACT GGT GGC 1010 Val Cys Asn GlyLeu Leu Gly Gly Phe Ala Ala Ile Thr Gly Gly TGC TCT GTC GTT GAG CCA TGGGCT GCG ATC ATC TGC GGG TTC GTG 1055 Cys Ser Val Val Glu Pro Trp Ala AlaIle Ile Cys Gly Phe Val GCG GCC CTA GTC CTC CTC GGA TGC AAC AAG CTC GCTGAG AAG CTC 1100 Ala Ala Leu Val Leu Leu Gly Cys Asn Lys Leu Ala Glu LysLeu AAA TAC GAC GAC CCT CTT GAG GCA GCA CAA CTA CAC GGT GGT TGC 1145 LysTyr Asp Asp Pro Leu G1u Ala Ala Gln Leu His Gly Gly Cys GGT GCG TGG GGACTA ATA TTC ACG GCT CTC TTC GCT CAA GAA AAG 1190 Gly Ala Trp Gly Leu IlePhe Thr Ala Leu Phe Ala Gln Glu Lys TAC TTG AAC CAG ATT TAC GGC AAC AAACCC GGA AGG CCA CAC GGT 1235 Tyr Leu Asn Gln Ile Tyr Gly Asn Lys Pro GlyArg Pro His Gly TTG TTT ATG GGC GGT GGA GGA AAA CTA CTT GGA GCT CAG CTGATT 1280 Leu Phe Met Gly Gly Gly Gly Lys Leu Leu Gly Ala Gln Leu Ile CAGATC ATT GTG ATC ACG GGT TGG GTA AGT GCG ACC ATG GGG ACA 1325 Gln Ile IleVal Ile Thr Gly Trp Val Ser Ala Thr Met Gly Thr CTT TTC TTC ATC CTC AAGAAA ATG AAA TTG TTG CGG ATA TCG TCC 1370 Leu Phe Phe Ile Leu Lys Lys MetLys Leu Leu Arg Ile Ser Ser GAG GAT GAG ATG GCC GGT ATG GAT ATG ACC AGGCAC GGT GGT TTT 1415 Glu Asp Glu Met Ala Gly Met Asp Met Thr Arg His GlyGly Phe GCT TAT ATG TAC TTT GAT CAT GAT GAG TCT CAC AAA GCC ATT CAG 1460Ala Tyr Met Tyr Phe Asp Asp Asp Glu Ser His Lys Ala Ile Gln CTT AGG AGAGTT GAG CCA CGA TCT CCT TCT CCT TCT GGT GCT AAT 1505 Leu Arg Arg Val GluPro Arg Ser Pro Ser Pro Ser Gly Ala Asn ACT ACA CCT ACT CCG GTTTGATTTGGAT TTTTACTTTT ATTCTCTATT 1553 Thr Thr Pro Thr Pro Val TTCTAGAGTATTATTTTAAA TGATGTTTTG TGATACTTAA ATATTGTTTT 1603 GGATATTTTT TTCGCATTTCAGTAATGTTT TAGATGTACA GTTTCATCGG 1653 GTTGTGATGA TAATATCTAT GTGGTCATTTGTGTTCTCTT TCGAGTTAAA 1703 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAAAAAAA 1748

[0035] The DNA sequences of the invention, identified using thetransformed yeast strain, such as e.g. the sequence Seq. ID No. 1, canbe introduced into plasmids and thereby be combined with regulatoryelements for expression in eukaryotic cells (see Example 4). Theseregulatory elements are on the one hand transcription promoters, and onthe other hand transcription terminators. With the plasmids, eukaryoticcells can be transformed, with the aim of expression of a translatablemRNA which makes possible the synthesis of an ammonium transporter inthe cells (sense orientation) or with the aim of expression of anon-translatable RNA, which prevents synthesis of an endogenous ammoniumtransporter in the cells (anti-sense orientation).

[0036] These plasmids are also an aspect of the invention.

[0037] Preferred plasmids are plasmids p35S-MEP-a and p35S-a-MEP-a,which have been deposited at the Deutsche Sammlung von Mikroorganismen(DSM).

[0038] A still further aspect of the present invention are yeasts, whichcontain the DNA sequences of the invention.

[0039] The yeast strains used for the identification of a plantmethylamine or ammonium transporter can be used for studies of theproperties of the transporter as well as its substrate. The DNAsequences of a plant ammonium transporter present in plasmids can, inaccordance with results of these studies, be subjected to a mutagenesisor sequence modification by recombination, in order to change theproperties of the transporter. By changing the specificity of thetransport system, the transport of new compounds is possible, whichopens up interesting applications (see below). In addition, by suitablechange of the transporter, the transport mechanism can be modified. Onecan envisage, especially but not exclusively, a change, that modifiesthe cotransport properties of the transporter, for example with theeffect that this results in an ammonium ion-proton-antiport, whichprevents a toxicity of ammonium ions taken up for plants as a result ofacidification of the cytoplasm. In this way, the yeast strain of theinvention, which contains the cDNA sequence of a plant ammoniumtransporter in an expression plasmid, can be used for a mutationselection system.

[0040] By expression of an RNA corresponding to the DNA sequences of theinvention of plant ammonium transporters in transgenic plants it ispossible to have a change of the plant nitrogen metabolism, whoseeconomic significance is obvious. Nitrogen is the nutrient mainlyresponsible for limiting growth. The viability of seedlings as well asgermination capacity of seeds is directly dependent on the nitrogencontent of storage tissue. The formation of high value food materialswith a high protein content is dependent on a sufficient nitrogensupply.

[0041] The change of nitrogen uptake, for example by addition of a newuptake system of nitrogen compounds such as ammonium, can thus lead toan increase in yield of transgenic crops, especially under nitrogenlimitation. In this way, transgenic plants can be cultivated in highyields under low input conditions.

[0042] The possibility of suppressing the take-up of ammonium in thetransgenic plants can however also be desirable under certainconditions. For example one can envisage the cultivation of transgenicplants on acid soils which are not suitable for growth. As a result ofsuppressing nitrification ammonium ion concentrations on such soilscould be present which would be toxic for certain plants, because, theammonium take up from the plants in the cells can no longer be fullymetabolised and acts as a cell poison. The suppression of the take-up ofammonium by suppressing the biosynthesis of the ammonium transporter canthus alter transgenic plants to the extent that cultivation on acidsoils becomes possible.

[0043] A further aspect of the present invention are transgenic plants,in which the DNA sequences of the invention are introduced as aconstituent of a recombinant DNA molecule, in which this recombinant DNAmolecule is stably integrated into the genome, and in whose cells basedon the presence of these sequences there is achieved either a synthesisof an additional plant ammonium transporter whereby these cells can takeup larger amounts of ammonium in comparison with untransformed plants orthere is achieved an inhibition of the synthesis of endogenous ammoniumtransporters whereby such cells show a reduced uptake of ammonium incomparison with untransformed cells.

[0044] Transgenic crops are, for example tobacco, potatoes, sugar beet,soya beans, peas, beans or maize.

[0045] The genetic modification of dicotyledonous and monocotyledonousplants can be carried out by currently known processes, (see for exampleGasser, C. S., Fraley, R. T., 1989, Science 244: 1293-1299; Potrykus,1991, Ann Rev Plant Mol Biol Plant Physiol 42: 205-225). For expressionin plants the coding sequences must be coupled with the transcriptionalregulatory elements. Such elements called promoters, are known (EP375091).

[0046] Further, the coding regions must be provided with transcriptiontermination signals with which they can be correctly transcribed. Suchelements are also described (see Gielen et al., 1989, EMBO J 8: 23-29).The transcriptional start region can be both native and/or homologous aswell as foreign and/or heterologous to the host plant. If desired,termination regions are interchangeable with one another. The DNAsequence of the transcription starting and termination regions can beprepared synthetically or obtained naturally, or obtained from a mixtureof synthetic and natural DNA constituents. For introduction of foreigngenes in higher plants a large number of cloning vectors are availablethat include a replication signal for E. coli and a marker which allowsa selection of the transformed cells. Examples of such vectors are pBR322, pUC-Series, M13 mp-Series, pACYC 184 etc. Depending on the methodof introduction of the desired gene in the plants, other DNA sequencesmay be suitable. Should the Ti- or Ri-plasmid be used, e.g. for thetransformation of the plant cell, then at least the right boundary,often however both the right and left boundary of the Ti- and Ri-PlasmidT-DNA, is attached, as a flanking region, to the gene being introduced.The use of T-DNA for the transformation of plants cells has beenintensively researched and is well described in EP 120 516; Hoekama, In:The Binary Plant Vector System, Offsetdrukkerij Kanters B. V.Alblasserdam, (1985), Chapter V; Fraley, et al., Crit. Rev. Plant Sci.,4:1-46 and An et al. (1985) EMBO J. 4: 277-287. Once the introduced DNAis integrated in the genome, it is as a rule stable there and remainsalso in the offspring of the original transformed cells. It normallycontains a selection marker, which induces resistance in the transformedplant cells against a biocide or antibiotic such as kanamycin, G 418,bleomycin, hygromycin or phosphinotricin etc. The individual markeremployed should therefore allow the selection of transformed cells fromcells, which lack the introduced DNA.

[0047] For the introduction of DNA into a plant host cell, besidestransformation using Agrobacteria, there are many other techniquesavailable. These techniques include the fusion of protoplasts,microinjection of DNA and electroporation, as well as ballistic methodsand virus infection. From the transformed plant material, whole plantscan be regenerated in a suitable medium, which contains antibiotics orbiocides for the selection. The resulting plants can then be tested forthe presence of introduced DNA.

[0048] A further aspect of the present invention are transformed plantcells, in which the DNA sequences of the invention are introduced as aconstituent of a recombinant DNA molecule, in which this recombinant DNAmolecule is stably integrated into the genome, and in whose cells basedon the presence of these sequences there is achieved either a synthesisof an additional plant ammonium transporter whereby these cells can takeup larger amounts of ammonium in comparison with untransformed plants orthere is achieved an inhibition of the synthesis of endogenous ammoniumtransporters whereby such cells show a reduced uptake of ammonium incomparison with untransformed cells.

[0049] No special demands are placed on the plasmids in injection andelectroporation. Simple plasmids, such as e.g. pUC-derivatives can beused. Should however whole plants be regenerated from such transformedcells the presence of a selectable marker gene is necessary. Thetransformed cells grow within the plants in the usual manner (see alsoMcCormick et al. (1986) Plant Cell Reports 5: 81-84). These plants canbe grown normally and crossed with plants, that possess the sametransformed genes or different. The resulting hybrid individuals havethe corresponding phenotypical properties.

[0050] The introduction of DNA plant ammonium transporters for changingthe uptake of nitrogen compounds is described here using Arabidopsisthaliana and tobacco as examples. The use is not however limited to thisplant species.

[0051] The DNA sequences of the invention can also be introduced inplasmids and thereby combined with steering elements for an expressionin prokaryotic cells. The formation of a translatable RNA sequence of aeukaryotic ammonium transporter from bacteria leads, in spite of theconsiderable differences in the membrane structures of prokaryotes andeukaryotes, means in addition surprisingly, that prokaryotes can nowexpress a functional eukaryotic ammonium transporter with its substratespecificity. This makes possible the production of bacterial strains,which as for the yeast strain used for identifying the ammoniumtransporter could be used for studies of the properties of thetransporter as well as its substrate, which opens up interestingapplications.

[0052] The invention also relates to bacteria, that contain the plasmidsof the invention.

[0053] The DNA sequences of the invention can also be introduced inplasmids which allow mutagenesis or a sequence modification throughrecombination of DNA sequences using standard microbiological processes.In this way the specificity of the ammonium transporter can be modified.

[0054] Modified ammonium transporters can be used for example fortransformation of agricultural transgenic plants, whereby both transportof pesticides and also plant growth regulators to plants can beenvisaged.

[0055] By using standard processes (see Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, 2. Edn., Cold Spring Harbor LaboratoryPress, NY, USA) base exchanges can be carried out or natural orsynthetic sequences can be added. For fusing DNA fragments with oneanother adaptors or linkers can be added to the fragments. Further,manipulations can be carried out which prepare suitable restrictioncleavage sides or remove the excess DNA or restriction cleavage sites.Where insertions, deletions or substitutions such as for exampletransitions and transversions are to be carried out, in vitromutagenesis, primer repair, restrictions or ligations can be used. Formethods of analysis, in general a sequence analysis, restrictionanalysis and other biochemical molecular biological methods can be used.After each manipulation, the DNA sequence, used, can be cleaved andfused with another DNA sequence. Each plasmid sequence can be cloned inthe same or different plasmids.

[0056] The invention also relates to derivatives or parts of plasmids onwhich the DNA sequences of the invention are localised.

[0057] Derivatives or parts of the DNA sequences and plasmids of theinvention can also be used for the transformation of prokaryotic andeukaryotic cells.

[0058] Further, the DNA sequences of the invention can be used accordingto standard processes for the isolation of homologous sequences from thegenome of plants of various species, which also code for ammmoniumtransporter molecules. With these sequences, constructs for thetransformation of plant cells can be prepared which modify the transportprocesses in transgenic plants.

[0059] By the terms “homology” or “homologous sequences” are to beunderstood, a sequence identity of 60% to 80%, preferably 80% to 95% andespecially 95% to 100%.

[0060] In order to specify related DNA sequences, gene libraries mustfirst be prepared, which are representative for the content in genes ofa plant species or for the expression of genes in a plant species. Theformer are genomic libraries, whilst the latter are cDNA libraries. Fromthese, related sequences can be isolated using the DNA sequences of theinvention as probes. Once the related gene has been identified andisolated, a determination of the sequence and an analysis of theproperties of the proteins coded from this sequence is possible.

[0061] DNA sequences of ammonium transporters obtained in this way arealso part of the invention and could be used as described above.

[0062] The use of the DNA sequences as described above is also part ofthe invention.

[0063] A further aspect of the invention are DNA sequences from plants,which hybridise with DNA sequences of the invention and code for aprotein that possesses the biological activity of an ammoniumtransporter. The term “hybridisation” means in this connection, ahybridisation under conventional hybridisation conditions, preferablyunder stringent conditions, such as described for example by Sambrook etal. (1989, Molecular Cloning, A Laboratory Manual, 2. Edn. Cold SpringHarbor Laboratory Press, Cold Spring Harbour, N.Y.). An importantbiological activity of an ammonium transporter is the capability oftransporting ammonium or analogues thereof through biological membranes.This activity can be measured by uptake of ammonium or analogues thereofby cells, which express the particular ammonium transporter, asdescribed for example in example 3 of the present invention.

[0064] Deposits

[0065] The following plasmids were deposited at the Deutschen Sammlungvon Mikroorganismen (DSM) in Braunschweig, Germany on the 26.10.1993(deposit number): Plasmid p35S-MEP-a (DSM 8651) Plasmid p35S-a-MEP-a(DSM 8652)

DESCRIPTION OF THE FIGURES

[0066]FIG. 1 shows the plasmid p35S-MEP-a, which is a derivative of theplasmids pBIN19 (Bevan, M., 1984, Nucl Acids Res 12: 8711-8721). Itcomprises:

[0067] A=Fragment from the genome of the cauliflower mosaic virus thatcarries the 35S promoter (nt 6909-7437). The promoter fragment wasprepared as an EcoRI/KpnI fragment from plasmid pDH51 (Pietrzak et al.,Nucl. Acid Res. 14, 5857-5868).

[0068] B=NotI/NotI fragment of the cDNA with the coding region of theammonium transporter of Arabidopsis thaliana in sense orientation to thefragment A. The arrow marked in fragment B indicates the readingdirection of the cDNA.

[0069] C=Polyadenylation signal of the gene 3 of the T-DNA of theplasmid pTiACH5 (Gielen et al., EMBO J 3: 835-846), nucleotides 11749 to11939, which was isolated as a PvuII/HindIII fragment from plasmidpAGV40 (Herrera-Estrella et al., Nature 303, 209-213) and, after theaddition of SphI linker onto the PvuII cleavage site, was cloned betweenthe SphI and HindIII cleavage sites of the polylinker from pBIN19.

[0070]FIG. 2 shows the plasmid p35S-a-MEP-a, which is a derivative ofthe plasmids pBIN19 (Bevan, M., 1984, Nucl Acids Res 12: 8711-8721). Itcomprises:

[0071] A=Fragment from the genome of the cauliflower mosaic virus thatcarries the 35S promoter (nt 6909-7437). The promoter fragment wasprepared as an EcoRI/KpnI fragment from plasmid pDH51 (Pietrzak et al.,Nucl. Acid Res. 14, 5857-5868).

[0072] B=NotI/NotI fragment of the cDNA with the coding region of theammonium transporter of Arabidopsis thaliana in anti-sense orientationto the fragment A. The arrow marked in fragment B indicates the readingdirection of the cDNA.

[0073] C=Polyadenylation signal of the gene 3 of the T-DNA of theplasmid pTiACH5 (Gielen et al., EMBO J 3: 835-846), nucleotides 11749 to11939, which was isolated as a PvuII/HindIII fragment from plasmidpAGV40 (Herrera-Estrella et al., Nature 303, 209-213) and, after theaddition of SphI linker onto the PvuII cleavage site, was cloned betweenthe SphI and HindIII cleavage sites of the polylinker from pBIN19.

[0074] The following examples illustrate the invention without limitingit in which there is described the cloning and identification as well asthe function of a plant ammonium transporter and its use for the geneticmodification of plants with aim of changing the uptake and transfer ofnitrogen compounds. In an analogous manner to the Examples, other plantsespecially crop plants such as potato, sugar beet, maize, etc, can bemodified.

[0075] In Examples 1 to 4 the following standard processes and specialtechniques were used.

[0076] 1. Cloning Process

[0077] For cloning in E. coli, a derivative of the vector pACYC thatcontains the polylinker from pBluescriptSK was used.

[0078] For the transformation of yeasts, the vector pFL61 (Minet &Lacroute, 1990,Curr Genet 18: 287-291) was used.

[0079] For the plant transformation the gene constructs were cloned inthe binary vector pBinAR, a derivative of pBIN19 (Bevan, 1984, NuclAcids Res 12: 8711-8721), was cloned (see Example 4).

[0080] 2. Bacterial and Yeast Strains

[0081] For the pACYC vector as well as for pBinAR constructs, E. colistrain DH5α was used.

[0082] As starting strain for the production of the cDNA library inyeast, the yeast strain 26972c (Dubois & Grenson, 1979, Mol Gen Gnete175: 67-76) with the mutations mep-1/mep-2 and ura3 was used.

[0083] The transformation of the plasmid in potato plants was carriedout using Agrobacterium tumefaciens strain LBA4404 (Bevan (1984) Nucl.Acids Res 12: 8711-8720).

[0084] 3. Transformation of Agrobacterium tumefaciens

[0085] The transfer of the DNA in the Agrobacteria was carried out bydirect transformation by the method of Hófgen & Willmitzer (1988,Nucleic Acids Res 16: 9877). The plasmid DNA of the transformedAgrobacterium was isolated in accordance with the method of Birnboim andDoly (1979) (Nucl Acids Res 7: 1513-1523) and was analysed by gelelectrophoresis after suitable restriction cleavage.

[0086] 4. Plant Transformation

[0087] Tobacco

[0088] 10 ml of an Agrobacterium tumefaciens overnight culture grownunder selection was sedimented and resuspended in the same volume ofantibiotic free medium. In a sterile petri dish, leaf discs of sterileplants (approximately 1 cm²), the central vein of which had beenremoved, were immersed in this bacterial suspension. The leaf discs werethen placed in a closely packed arrangement in petri dishes containingMS medium (Murashige et al. (1962) Physiologia Plantarum 15, 473-497)with 2% sucrose and 0.8% bacto agar. After two days incubation in thedark at 25° C., they were transferred onto MS medium containing 500 mg/lClaforan, 50 mg/l kanamycin, 1 mg/l benzylaminopurine (BAP), 0.2 mg/l ofnaphthylacetic acid (NAA) and 0.8% bacto agar. Growing shoots weretransferred onto hormone-free MS medium with 250 mg/l Claforan and 50mg/l Kanamycin.

[0089]Arabidopsis thaliana

[0090] Cotyledons from a seven day old sterile culture of Arabidopsisthaliana grown from seeds, were pre-incubated on Cl-Medium for two dayswith a layer of tobacco suspension culture (as feeder layer) and thenfor 5 minutes in a suspension with Agrobacterium tumefaciens(preparation of suspension see under tobacco transformation) and thenincubated for two days on Cl-Medium with feeder layer in low lightconditions. After this cocultivation with Agrobakteria the explantateswere laid out on SIM I-medium, that was weekly renewed. Aftercallous-formation the explantates were set transplanted to SIM II-mediumand the cultivated under selection with kanamycin. SmaII shoots wereseparated from the callous material and transplanted to RI-Medium. Afterseparation of seeds, these could be analysed.

[0091] The individual media were as follows: Cl-medium SIM I-medium SIMII-medium RI-Medium Murashige- Murashige- Murashige- Murashige- Skoogmedium Skoog medium Skoog medium Skoog with 2% with 2% with 2% mediumwith sucrose, 8 g/l sucrose, 8 g/l sucrose, 8 g/l 1% agar, agar, agar,sucrose, 1 mg/l 2,4- 1 mg/l 6-benzyl- 7 mg/l 8 g/l agar, dichloro-aminopurine, 0.4 N⁶-[2-iso- 1 mg/l phenoxy-acetic mg/l pentenyl]-indolyl- acid (2,4-D), naphthyl-acetic adenine, 0.05 butyric 0.2 mg/lkinetin acid, 0.5 g/l mg/l indolyl-acetic acid Claforan, acid, 0.5 mg/l100 mg/l Clatoran, kanamycin 100 mg/l kanamycin

EXAMPLE 1

[0092] Cloning of the cDNA of a Plant Methylamine or AmmoniumTransporter

[0093] For complementation of the ammonium transport double mutation ofthe yeast strain 26972c (Dubois & Grenson, 1979, Mol Gen Genet 175:67-76), there was used a cDNA of young germ lines from Arabidopsisthaliana (two leaf stage) in the yeast expression vector pFL61 (Minet &Lacroute, 1990, Curr Genet 18: 287-291) which had been made available byMinet (Minet et al., 1992, Plant J 2: 417-422). Around 1 μg of thevector with the cDNA-insert was transformed in the yeast strain 26972cby the method of Dohmen et al. (1991, Yeast 7: 691-692). Yeasttransformands, which could grow in minimal medium with 1 mM NH₄Cl as thesole nitrogen source were propagated. From the lines, plasmid-DNA wasprepared by standard methods. This DNA was immediately transformed instrain 26972c. In this way a plasmid pFL61-MEP-a was obtained that cancomplement the mep-1/mep-2 double mutation. This plasmid has aninsertion of size 1.75 kbp.

[0094] The yeast strain 26972c::pFL61-MEP-a obtain by transformation of26972c with plasmid pFL61-MEP-a can be used for uptake studies withmethylamine or ammonium (see example 3). By genetic modification of thecoding region of the ammonium transporter gene MEP-a by standard methods(cf. Sambrook et al., 1989, Molecular cloning: A laboratory manual, 2.Edn., Cold Spring Harbor Laboratory Press, NY, USA) the specificity orcharacteristic of the transport mechanism of which can be modified. Thestrain 26972c::pFL61-MEP-a is suitable directly for testing forinhibitors or promoters of the ammonium transport, using the ammoniumtransport system of the invention (see Example 3).The cDNA insertion ofthe plasmids pFL61-MEP-a can be used for the identification of similarDNA sequences from plants of other species or from other organisms, suchas bacteria or animal systems. For this hybridisation techniques areusable. Likewise using the cDNA sequence, a construct for the expressionof the gene product as a fusion protein in bacteria can be prepared bystandard processes. Using the fusion protein, an antibody, whichidentifies similar proteins in other organisms, can be prepared.

EXAMPLE 2

[0095] Sequence Analyses of the cDNA Insert of the Plasmid pFL61-MEP-a

[0096] From a yeast line 26972c::pFL61-MEP-a, obtained from example 1,the plasmid pFL61-MEP-a was isolated and its cDNA insert prepared as aNotI fragment. The fragment was cloned in a modified pACYC vector (Chang& Cohen, 1978, J Bacteriol 134. 1141-1156), which contained, between aHindII (nt 3211) and a filled HindIII (nt 1523) cutting position, thepolylinker from pBluescript as BssHII fragment. Through the modificationthe tetracycline resistance is lost. The cDNA fragment was cloned in thethus obtained vector pACH-H as NotI fragment in the NotI cuttingposition. Using synthetic oligonucleotides, the insert was sequenced bythe method of Sanger et al. (1977, Proc Natl Acad Sci USA 74:5463-5467). The sequence is given in Seq ID No 1.

EXAMPLE 3

[0097] Uptake Studies With ¹⁴C-labelled Methylamine Into the Yeast Line26972c::pFL61-MEP-a

[0098] The yeast lines 26972c::pFL61, 26972c::pFL61-MEP-a and theirstarting lines S1278b (Dubois & Grenson, 1979, Mol Gen Genet 175: 67-76,MEP-1/MEP-2 ura3) were grown in liquid medium with 1.7 g yeast nitrogenbase without ammonium and without amino acids (Difco), 20 g agarose, 1%glucose (w/v), 0.5 mg/l proline until the culture reached thelogarithmic phase. After centrifuging of each 25 ml of the culture thecells were washed with 10 mM phosphate pH 7 or pH 4 and taken up in 1.5ml 10 mM phosphate buffer. Ten minutes before starting the transportmeasurements, it was made up to an end concentration of 10 mM glucose.100 μml of the suspension was added to a solution of 10 mM phosphate pH4 or 7, 100 μM unlabelled methylamine and 5 μCi ¹⁴C-labelled methylamine(0.1 μCi/1.9 μmol) (end concentrations). The uptake of the methylaminewas measured after 10, 60, 120 and 180 seconds. For this, to 50 μl ofstarting material was taken and in a volume of 1 ml H₂O+3 mM unlabelledmethylamine absorbed on a glass fibre filter. After washing with 10 mlH₂O+3 mM unlabelled methylamine the amount of ¹⁴C-labelled methylaminetaken up was measured by scintillation (Table Ia and Ib). The uptake ofthe labelled methylamine was compared for the case of coincubation with0.5 mM NH₄Cl (Table II) and 0.5 mM KCl (Table III), as well as for thecase of coincubation with the uncouplers, dinitrophenol (DNP) andcarbonyl cyanide m-chlorophenylhydrazone (CCCP) (Table IV). The valuesof a typical experiment are given in Tables I to IV: TABLE 1 Uptake of¹⁴C-methylamine in yeast strains after incubation with 10 mM glucose atdifferent pH values (at each time-point for the MEP-a transformands andthe zero controls (plasmid pFL61 without cDNA insertion) values from twoindependent series of measurements are given. Measurements in cpm(counts per minute) Time point [sec] Σ1278b 26972c::pFL6126972c::pFL61-MEP-a pH = 4  10 289.00 220.20 117.20 204.80 214.80  601151.20 200.60 138.60 283.80 345.60 120 19605.46 284.80 197.40 434.40594.60 180 32651.43 317.60 285.60 479.60 842.20 pH = 7  10 469.60 202.20./. 302.80 352.20  60 1022.80 291.80 ./. 1247.80 1292.20 120 6176.36524.80 ./. 3152.50 3478.28 180 16826.67 797.80 ./. 8490.83 6099.39

[0099] TABLE II Uptake of ¹⁴C-methylamine in yeast strains afterincubation with 10 mM glucose in presence of 0.5 mM NH₄Cl. Measurementsin cpm. pH = 7 Time point [sec] 1278b 26972c::pFL61 26972c::pFL61-MEP-a 10 356.60 271.60 268.60  60 885.60 795.80 577.60 120 1237.80 1388.20978.00 180 1597.40 1773.00 1226.60

[0100] TABLE III Uptake of ¹⁴C-methylamine in yeast strains afterincubation with 10 mM glucose in presence of 0.5 mM KCl, unlessotherwise noted. Measurements in cpm. pH = 7 Time 26972c::pFL61- point26972c::pFL6l- MEP-a without [sec] Σ1278b 26972c::pFL61 MEP-aKCl-competition 10 391.20 127.60 384.00 205.00 60 2054.90 224.00 1276.60467.60 120 8455.00 374.80 3128.44 1541.00 180 14377.14 567.40 5056.002416.39

[0101] TABLE IV Uptake of ¹⁴C-methylamine in yeast strains afterincubation with 10 mM glucose in presence of 0.1 mM dinitrophenol (DNP)or 10 μM carbonyl cyanide m-chlorophenylhydraZone. Measurements in cpm(counts per minute). pH = 7 Time 26972c::pFL61- 26972c::pFl61 26972c::point Σ1278b Σ1278b MEP-a MEP-a pFl6- [sec] +DNP +CCCP +DNP +CCCP MEP-a10 167.20 173.20 378.00 484.60 373.60 60 386.00 316.80 1349.40 1407.001214.20 120 706.80 460.40 2794.44 2597.92 3673.45 180 906.00 608.002794.44 3620.36 6135.15

EXAMPLE 4

[0102] Transformation of Plants With a construct for Over-Expression ofthe Coding Region of the Ammonium Transporter

[0103] From the plasmid pFL61-MEP-a, that contains as insert the cDNAfor the methylamine or ammonium transporter from Arabidopsis thaliana,the insert was isolated as NotI fragment and cloned after filling in theoverhanging ends in the SmaI cutting position of pBinAR. The cDNA hasthe designation “B” in the plasmid map (FIG. 1). According to whether ornot B is introduced in sense orientation to the 35S Promoter of pBinAR,the resulting plasmid has the designation p35S-MEP-a or p35S-a-MEP-a.The plasmid pBinAR is a derivative of pBIN19 (Bevan, 1984, Nucl AcidsRes 12: 8711-8720). Between its EcoRI and KpnI cutting positions, afragment from the genome of the cauliflower mosaic virus, that carriesthe 35S promoter (nt 6909-7437), was introduced. The promoter fragmentprepared as EcoRI/KpnI fragment from the plasmid pDH51 (Pietrzak et al.,Nucl Acids Res 14: 5857-5868). In the plasmid map the promoter fragmenthas the designation “A”. Between the SphI and the HindIII cuttingposition of pBinAR, the polyadenylation signal of the gene 3 of theT-DNA of the plasmid pTiACH5 (Gielen et al., EMBO J 3:835-846) isinserted. Also a PvuII/HindIII fragment (nt 11749-11939) from theplasmid pAGV 40 (Herrera-Estrella et al., 1983, Nature 303: 209-2139)was supplied at the PvuII cutting position with a SphI linker. Thepolyadenylation signal has the designation “C” in the plasmid map.

[0104] After transformation of Agrobacteria with the plasmids p35S-MEP-aand p35S-a-MEP-a, these were used for infection of leaf segments oftobacco and Arabidopsis thaliana.

[0105] Ten independently obtained transformands for both constructs, inwhich the presence of the intact, non-rearranged chimeric gene wasdemonstrated using Southern blot analysis, were tested for changes innitrogen content.

0 SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 2(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 1748 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: (A)ORGANISM: Arabidopsis thaliana (C) INDIVIDUAL ISOLATE: Ammoniumtransporter (vii) IMMEDIATE SOURCE: (A) LIBRARY: cDNA library in plasmidpF161 (viii) POSITION IN GENOME: (B) MAP POSITION: from 21 to 1523coding region (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 21..1526(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TTCTTCTCTA AACTCTCAAC ATG TCTTGC TCG GCC ACC GAT CTC GCC GTC 50 Met Ser Cys Ser Ala Thr Asp Leu AlaVal 1 5 10 CTG TTG GGT CCT AAT GCC ACG GCG GCG GCC AAC TAC ATA TGT GGCCAG 98 Leu Leu Gly Pro Asn Ala Thr Ala Ala Ala Asn Tyr Ile Cys Gly Gln15 20 25 CTA GGC GAC GTC AAC AAC AAA TTC ATC GAC ACC GCT TTC GCT ATA GAC146 Leu Gly Asp Val Asn Asn Lys Phe Ile Asp Thr Ala Phe Ala Ile Asp 3035 40 AAC ACT TAC CTC CTC TTC TCC GCC TAC CTT GTC TTC TCT ATG CAG CTT194 Asn Thr Tyr Leu Leu Phe Ser Ala Tyr Leu Val Phe Ser Met Gln Leu 4550 55 GGC TTC GCT ATG CTC TGT GCC GGT TCC GTG AGA GCC AAG AAT ACT ATG242 Gly Phe Ala Met Leu Cys Ala Gly Ser Val Arg Ala Lys Asn Thr Met 6065 70 AAC ATC ATG CTT ACC AAC GTC CTT GAC GCT GCA GCC GGT GGT CTC TTC290 Asn Ile Met Leu Thr Asn Val Leu Asp Ala Ala Ala Gly Gly Leu Phe 7580 85 90 TAT TAT CTG TTT GGC TAC GCC TTT GCC TTT GGA TCT CCG TCC AAT GGT338 Tyr Tyr Leu Phe Gly Tyr Ala Phe Ala Phe Gly Ser Pro Ser Asn Gly 95100 105 TTC ATC GGT AAA CAC TAC TTT GGT CTC AAA GAC ATC CCC ACG GCC TCT386 Phe Ile Gly Lys His Tyr Phe Gly Leu Lys Asp Ile Pro Thr Ala Ser 110115 120 GCT GAC TAC TCC AAC TTT CTC TAC CAA TGG GCC TTT GCA ATC GCT GCG434 Ala Asp Tyr Ser Asn Phe Leu Tyr Gln Trp Ala Phe Ala Ile Ala Ala 125130 135 GCT GGA ATC ACA AGT GGC TCG ATC GCT GAA CGG ACA CAG TTC GTG GCT482 Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr Gln Phe Val Ala 140145 150 TAC CTA ATC TAT TCC TCT TTC TTA ACC GGG TTT GTT TAC CCG GTC GTC530 Tyr Leu Ile Tyr Ser Ser Phe Leu Thr Gly Phe Val Tyr Pro Val Val 155160 165 170 TCT CAC TGG TTC TGG TCA GTT GAT GGA TGG GCC AGC CCG TTC CGTACC 578 Ser His Trp Phe Trp Ser Val Asp Gly Trp Ala Ser Pro Phe Arg Thr175 180 185 GAT GGA GAT TTG CTT TTC AGC ACC GGA GCG ATA GAT TTC GCT GGGTCC 626 Asp Gly Asp Leu Leu Phe Ser Thr Gly Ala Ile Asp Phe Ala Gly Ser190 195 200 GGT GTT GTT CAT ATG GTC GGA GGT ATC GCT GGA CTC TGG GGT GCGCTC 674 Gly Val Val His Met Val Gly Gly Ile Ala Gly Leu Trp Gly Ala Leu205 210 215 ATC GAA GGT CCA CGA CTT GGC CGG TTC GAT AAC GGA GGC CGT GCCATC 722 Ile Glu Gly Pro Arg Leu Gly Arg Phe Asp Asn Gly Gly Arg Ala Ile220 225 230 GCT CTT CGT GGC CAC TCG GCG TCA CTT GTT GTC CTT GGA ACA TTCCTC 770 Ala Leu Arg Gly His Ser Ala Ser Leu Val Val Leu Gly Thr Phe Leu235 240 245 250 CTC TGG TTT GGA TGG TAC GGA TTT AAC CCC GGT TCC TTC AACAAG ATC 818 Leu Trp Phe Gly Trp Tyr Gly Phe Asn Pro Gly Ser Phe Asn LysIle 255 260 265 CTA GTC ACG TAC GAG ACA GGC ACA TAC AAC GGC CAG TGG AGCGCG GTC 866 Leu Val Thr Tyr Glu Thr Gly Thr Tyr Asn Gly Gln Trp Ser AlaVal 270 275 280 GGA CGG ACA GCT GTC ACA ACA ACG TTA GCT GGC TGC ACC GCGGCG CTG 914 Gly Arg Thr Ala Val Thr Thr Thr Leu Ala Gly Cys Thr Ala AlaLeu 285 290 295 ACA ACC CTA TTT GGG AAA CGT CTA CTC TCG GGA CAT TGG AACGTC ACT 962 Thr Thr Leu Phe Gly Lys Arg Leu Leu Ser Gly His Trp Asn ValThr 300 305 310 GAT GTA TGC AAC GGC CTC CTC GGA GGG TTT GCA GCC ATA ACTGGT GGC 1010 Asp Val Cys Asn Gly Leu Leu Gly Gly Phe Ala Ala Ile Thr GlyGly 315 320 325 330 TGC TCT GTC GTT GAG CCA TGG GCT GCG ATC ATC TGC GGGTTC GTG GCG 1058 Cys Ser Val Val Glu Pro Trp Ala Ala Ile Ile Cys Gly PheVal Ala 335 340 345 GCC CTA GTC CTC CTC GGA TGC AAC AAG CTC GCT GAG AAGCTC AAA TAC 1106 Ala Leu Val Leu Leu Gly Cys Asn Lys Leu Ala Glu Lys LeuLys Tyr 350 355 360 GAC GAC CCT CTT GAG GCA GCA CAA CTA CAC GGT GGT TGCGGT GCG TGG 1154 Asp Asp Pro Leu Glu Ala Ala Gln Leu His Gly Gly Cys GlyAla Trp 365 370 375 GGA CTA ATA TTC ACG GCT CTC TTC GCT CAA GAA AAG TACTTG AAC CAG 1202 Gly Leu Ile Phe Thr Ala Leu Phe Ala Gln Glu Lys Tyr LeuAsn Gln 380 385 390 ATT TAC GGC AAC AAA CCC GGA AGG CCA CAC GGT TTG TTTATG GGC GGT 1250 Ile Tyr Gly Asn Lys Pro Gly Arg Pro His Gly Leu Phe MetGly Gly 395 400 405 410 GGA GGA AAA CTA CTT GGA GCT CAG CTG ATT CAG ATCATT GTG ATC ACG 1298 Gly Gly Lys Leu Leu Gly Ala Gln Leu Ile Gln Ile IleVal Ile Thr 415 420 425 GGT TGG GTA AGT GCG ACC ATG GGG ACA CTT TTC TTCATC CTC AAG AAA 1346 Gly Trp Val Ser Ala Thr Met Gly Thr Leu Phe Phe IleLeu Lys Lys 430 435 440 ATG AAA TTG TTG CGG ATA TCG TCC GAG GAT GAG ATGGCC GGT ATG GAT 1394 Met Lys Leu Leu Arg Ile Ser Ser Glu Asp Glu Met AlaGly Met Asp 445 450 455 ATG ACC AGG CAC GGT GGT TTT GCT TAT ATG TAC TTTGAT GAT GAT GAG 1442 Met Thr Arg His Gly Gly Phe Ala Tyr Met Tyr Phe AspAsp Asp Glu 460 465 470 TCT CAC AAA GCC ATT CAG CTT AGG AGA GTT GAG CCACGA TCT CCT TCT 1490 Ser His Lys Ala Ile Gln Leu Arg Arg Val Glu Pro ArgSer Pro Ser 475 480 485 490 CCT TCT GGT GCT AAT ACT ACA CCT ACT CCG GTTTGA TTTGGATTTT 1536 Pro Ser Gly Ala Asn Thr Thr Pro Thr Pro Val * 495500 TACTTTTATT CTCTATTTTC TAGAGTATTA TTTTAAATGA TGTTTTGTGA TACTTAAATA1596 TTGTTTTGGA TATTTTTTTG GCATTTCAGT AATGTTTTAG ATGTACAGTT TCATGGGGTT1656 GTGATGATAA TATCTATGTG GTCATTTGTG TTCTCTTTGG AGTTAAAAAA AAAAAAAAAA1716 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 1748 (2) INFORMATION FOR SEQ IDNO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 501 amino acids (B)TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Ser Cys Ser Ala Thr Asp Leu AlaVal Leu Leu Gly Pro Asn Ala 1 5 10 15 Thr Ala Ala Ala Asn Tyr Ile CysGly Gln Leu Gly Asp Val Asn Asn 20 25 30 Lys Phe Ile Asp Thr Ala Phe AlaIle Asp Asn Thr Tyr Leu Leu Phe 35 40 45 Ser Ala Tyr Leu Val Phe Ser MetGln Leu Gly Phe Ala Met Leu Cys 50 55 60 Ala Gly Ser Val Arg Ala Lys AsnThr Met Asn Ile Met Leu Thr Asn 65 70 75 80 Val Leu Asp Ala Ala Ala GlyGly Leu Phe Tyr Tyr Leu Phe Gly Tyr 85 90 95 Ala Phe Ala Phe Gly Ser ProSer Asn Gly Phe Ile Gly Lys His Tyr 100 105 110 Phe Gly Leu Lys Asp IlePro Thr Ala Ser Ala Asp Tyr Ser Asn Phe 115 120 125 Leu Tyr Gln Trp AlaPhe Ala Ile Ala Ala Ala Gly Ile Thr Ser Gly 130 135 140 Ser Ile Ala GluArg Thr Gln Phe Val Ala Tyr Leu Ile Tyr Ser Ser 145 150 155 160 Phe LeuThr Gly Phe Val Tyr Pro Val Val Ser His Trp Phe Trp Ser 165 170 175 ValAsp Gly Trp Ala Ser Pro Phe Arg Thr Asp Gly Asp Leu Leu Phe 180 185 190Ser Thr Gly Ala Ile Asp Phe Ala Gly Ser Gly Val Val His Met Val 195 200205 Gly Gly Ile Ala Gly Leu Trp Gly Ala Leu Ile Glu Gly Pro Arg Leu 210215 220 Gly Arg Phe Asp Asn Gly Gly Arg Ala Ile Ala Leu Arg Gly His Ser225 230 235 240 Ala Ser Leu Val Val Leu Gly Thr Phe Leu Leu Trp Phe GlyTrp Tyr 245 250 255 Gly Phe Asn Pro Gly Ser Phe Asn Lys Ile Leu Val ThrTyr Glu Thr 260 265 270 Gly Thr Tyr Asn Gly Gln Trp Ser Ala Val Gly ArgThr Ala Val Thr 275 280 285 Thr Thr Leu Ala Gly Cys Thr Ala Ala Leu ThrThr Leu Phe Gly Lys 290 295 300 Arg Leu Leu Ser Gly His Trp Asn Val ThrAsp Val Cys Asn Gly Leu 305 310 315 320 Leu Gly Gly Phe Ala Ala Ile ThrGly Gly Cys Ser Val Val Glu Pro 325 330 335 Trp Ala Ala Ile Ile Cys GlyPhe Val Ala Ala Leu Val Leu Leu Gly 340 345 350 Cys Asn Lys Leu Ala GluLys Leu Lys Tyr Asp Asp Pro Leu Glu Ala 355 360 365 Ala Gln Leu His GlyGly Cys Gly Ala Trp Gly Leu Ile Phe Thr Ala 370 375 380 Leu Phe Ala GlnGlu Lys Tyr Leu Asn Gln Ile Tyr Gly Asn Lys Pro 385 390 395 400 Gly ArgPro His Gly Leu Phe Met Gly Gly Gly Gly Lys Leu Leu Gly 405 410 415 AlaGln Leu Ile Gln Ile Ile Val Ile Thr Gly Trp Val Ser Ala Thr 420 425 430Met Gly Thr Leu Phe Phe Ile Leu Lys Lys Met Lys Leu Leu Arg Ile 435 440445 Ser Ser Glu Asp Glu Met Ala Gly Met Asp Met Thr Arg His Gly Gly 450455 460 Phe Ala Tyr Met Tyr Phe Asp Asp Asp Glu Ser His Lys Ala Ile Gln465 470 475 480 Leu Arg Arg Val Glu Pro Arg Ser Pro Ser Pro Ser Gly AlaAsn Thr 485 490 495 Thr Pro Thr Pro Val 500

1. DNA sequences that contain the coding region of a plant ammoniumtransporter, characterised in that the information contained in thenucleotide sequence when integrated in a plant genome makes possibleunder control of a promoter in sense orientation, the expression of atranslatable mRNA, which leads to the synthesis of an ammoniumtransporter in transgenic plants.
 2. DNA sequences, that contain thecoding region of a plant ammonium transporter, characterised in that theinformation contained in the nucleotide sequence when integrated in aplant genome makes possible under control of a promoter in anti-senseorientation, the expression of a non-translatable mRNA, which preventsthe synthesis of an endogenous ammonium transporter in transgenicplants.
 3. A DNA sequence according to claim 1 or 2, characterised inthat, it contains the following nucleotide sequence (Seq-ID No 1):                        TTGTTCTGTAAACTCTCAAC 20 ATG TCT TGC TGG GCC ACCGAT CTC GCC GTC CTG TTG GGT CCT AAT 65 Net Ser Gys Ser Ala Thr Asp LeuAla Val Leu Leu Gly Pro Asn GCC ACG GCG GCG GCC AAC TAC ATA TGT GGC CAGCTA GGC GAC GTC 110 Ala Thr Ala Ala Ala Asn Tyr Ile Cys Gly Gln Leu GlyAsp Val AAC AAC AAA TTC ATC GAC ACC GCT TTC GCT ATA GAC AAC ACT TAC 155Asn Asn Lys Phe Ile Asp Thr Ala Phe Ala Ile Asp Asn Thr Tyr CTC CTC TTCTCC GCC TAC CTT GTC TTC TCT ATG CAG CTT GGC TTC 200 Leu Leu Phe Ser AlaTyr Leu Val Phe Ser Met GIn Leu Gly Phe GCT ATG CTC TGT GCC CGT TCC GTGAGA GCC AAG AAT ACT ATG AAC 245 Ala Met Leu Cys Ala Gly Ser Val Arg AlaLys Asn Thr Met Asn ATC ATG CTT ACC AAC GTC CTT GAC GCT GCA GCC GGT GGTCTC TTC 290 Ile Met Leu Thr Asn Val Leu Asp Ala Ala Ala Gly Gly Leu PheTAT TAT CTG TTT GGC TAC GCC TTT GCC TTT GGA TCT CCG TCC AAT 335 Tyr TyrLeu Phe Gly Tyr Ala Phe Ala Phe Gly Ser Pro Ser Asn GGT TTC ATC GGT AAACAC TAC TTT GGT CTC AAA GAC ATC CCC ACG 380 Gly Phe Ile Gly Lys His TyrPhe Gly Leu Lys Asp Ile Pro Thr GCC TCT GCT GAC TAC TCC AAC TTT CTC TACCAA TGG GCC TTT GCA 425 Ala Ser Ala Asp Tyr Ser Asn Phe Leu Tyr Gln TrpAla Phe Ala ATC GCT GCG GCT GGA ATC ACA AGT GGC TCG ATC GCT GAA CGG ACA470 Ile Ala Ala Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr CAG TTCGTG GCT TAC CTA ATC TAT TCC TCT TTC TTA ACC GGG TTT 515 Gln Phe Val AlaTyr Leu Ile Tyr Ser Ser Phe Leu Thr Gly Phe GTT TAC CCG GTC GTC TCT CACTGG TTC TGG TCA GTT GAT GGA TGG 560 Val Tyr Pro Val Val Ser His Trp PheTrp Ser Val Asp Gly Trp GCC AGC CCG TTC CGT ACC GAT GGA GAT TTG CTT TTCAGC ACC GGA 605 Ala Ser Pro Phe Arg Thr Asp Gly Asp Leu Leu Phe Ser ThrGly GCG ATA GAT TTC GCT GGG TCC GGT GTT GTT CAT ATG GTC GGA GGT 650 AlaIle Asp Phe Ala Gly Ser Gly Val Val His Met Val Gly Gly ATC GCT GGA CTCTGG GGT GCG CTC ATC GAA GGT CCA CGA CTT GGC 695 Ile Ala Gly Leu Trp GlyAla Leu Ile Glu Gly Pro Arg Leu Gly CGG TTC GAT AAC GGA GGC CGT GCC ATCGCT CTT CGT GGC CAC TCG 740 Arg Phe Asp Asn Gly Gly Arg Ala Ile Ala LeuArg Gly His Ser GCG TCA CTT GTT GTC CTT GGA ACA TTC CTC CTC TGG TTT GGATGG 785 Ala Ser Leu Val Val Leu Gly Thr Phe Leu Leu Trp Phe Gly Trp TACGGA TTT AAC CCC GGT TCC TTC AAC AAG ATC CTA GTC ACG TAC 830 Tyr Gly PheAsn Pro Gly Ser Phe Asn Lys Ile Leu Val Thr Tyr GAG ACA GGC ACA TAC AACGGC CAG TGG AGC GCG GTC GGA CGG ACA 875 Glu Thr Gly Thr Tyr Asn Gly GlnTrp Ser Ala Val Gly Arg Thr GCT GTC ACA ACA ACG TTA GCT GGC TGC ACC GCGGCG CTG ACA ACC 920 Ala Val Thr Thr Thr Leu Ala Gly Cys Thr Ala Ala LeuThr Thr CTA TTT GGG AAA CGT CTA CTC TCG GGA CAT TGG AAC GTC ACT GAT 965Leu Phe Gly Lys Arg Leu Leu Ser Gly His Trp Asn Val Thr Asp GTA TGC AACGGC CTC CTC GGA GGG TTT GCA GCC ATA ACT GGT GGC 1010 Val Cys Asn Gly LeuLeu Gly Gly Phe Ala Ala Ile Thr Gly Gly TGC TCT GTC GTT GAG CCA TGG GCTGCG ATC ATC TGC GGG TTC GTG 1055 Cys Ser Val Val Glu Pro Trp Ala Ala IleIle Cys Gly Phe Val GCG GCC CTA GTC CTC CTC GGA TGC AAC AAG CTC GCT GAGAAG CTC 1100 Ala Ala Leu Val Leu Leu Gly Cys Asn Lys Leu Ala Glu Lys LeuAAA TAC GAC GAC CCT CTT GAG GCA GCA CAA CTA CAC GGT GGT TGC 1145 Lys TyrAsp Asp Pro Leu Glu Ala Ala Gln Leu His Gly Gly Cys GCT GCG TGG GGA CTAATA TTC ACG GCT CTC TTC GCT CAA GAA AAG 1190 Gly Ala Trp Gly Leu Ile PheThr Ala Leu Phe Ala Gln Glu Lys TAC TTG AAC CAG ATT TAC GGC AAC AAA CCCGGA AGG CCA CAC GGT 1235 Tyr Leu Asn Gln Ile Tyr Gly Asn Lys Pro Gly ArgPro His Gly TTG TTT ATG GGC GGT GGA GGA AAA CTA CTT GGA GCT CAG CTG ATT1280 Leu Phe Met Gly Gly Gly Gly Lys Leu Leu Gly Ala Gln Leu Ile CAG ATCATT GTG ATC ACG GGT TGG GTA ACT GCG ACC ATG GGG ACA 1325 Gln Ile Ile ValIle Thr Gly Trp Val Ser Ala Thr Met Gly Thr CTT TTC TTC ATC CTC AAG AAAATG AAA TTG TTG CGG ATA TCG TCC 1370 Leu Phe Phe Ile Leu Lys Lys Met LysLeu Leu Arg Ile Ser Ser GAG GAT GAG ATG GCC GGT ATG GAT ATG ACC AGG CACGGT GGT TTT 1415 Glu Asp Glu Met Ala Gly Met Asp Met Thr Arg His Gly GlyPhe GCT TAT ATG TAC TTT GAT GAT CAT GAG TCT CAC AAA GCC ATT CAG 1460 AlaTyr Met Tyr Phe Asp Asp Asp Glu Ser His Lys Ala Ile Gln CTT AGG AGA GTTGAG CCA CGA TCT CCT TCT CCT TCT GGT GCT AAT 1505 Leu Arg Arg Val Glu ProArg Ser Pro Ser Pro Ser Gly Ala Asn ACT ACA CCT ACT CCG GTT TGATTTGGATTTTTACTTTT ATTCTCTATT 1553 Thr Thr Pro Thr Pro Val TTCTAGAGTA TTATTTTAAATGATGTTTTG TGATACTTAA ATATTGTTTT 1603 GGATATTTTT TTGGCATTTC AGTAATGTTTTAGATGTACA GTTTCATGGG 1653 GTTGTGATGA TAATATCTAT GTGGTCATTT GTGTTCTCTTTGGAGTTAAA 1703 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA 1748


4. A process for the identification and isolation of DNA sequences fromplants that code for ammonium transporters, characterised in that itincludes the following steps: a) transformation of a yeast strain, whichcannot grow in media which contain ammonium as the only nitrogen source,with a plant cDNA-library using suitable expression vectors, b)selection and propagation of transformands, which after expression ofplant cDNA-sequences, can grow in media which contain ammonium as theonly nitrogen source, and c) isolation of the expression vectors, whichcarry a plant cDNA insert from the selected transformand.
 5. DNAsequences from plants, that code for a protein with the biologicalactivity of an ammonium transporter, obtainable by a process accordingto claim
 4. 6. Plasmids, containing DNA sequences according to any oneof claims 1-3.
 7. Plasmid p35S-MEP-a (DSM 8651).
 8. Plasmid p35S-a-MEP-a(DSM 8652).
 9. Use of the plasmid according to any one of claims 6-8 orderivatives or parts thereof for the transformation of pro- andeukaryotic cells.
 10. Transgenic plants, in which the DNA sequences ofthe invention are introduced as a constituent of a recombinant DNAmolecule, in which this recombinant DNA molecule is stably integratedinto the genome, and in whose cells based on the presence of thesesequences there is achieved either a synthesis of an additional plantammonium transporter whereby these cells can take up larger amounts ofammonium in comparison with untransformed plants or there is achieved aninhibition of the synthesis of endogenous ammonium transporters wherebysuch cells show a reduced uptake of ammonium in comparison withuntransformed cells
 11. Yeasts, containing DNA sequences according toany one of claims 1-3.
 12. Bacteria, containing DNA sequences accordingto any one of claims 1-3.
 13. Plant cells, in which the DNA sequences ofthe invention are introduced as a constituent of a recombinant DNAmolecule, in which this recombinant DNA molecule is stably integratedinto the genome, and in whose cells based on the presence of thesesequences there is achieved either a synthesis of an additional plantammonium transporter whereby these cells can take up larger amounts ofammonium in comparison with untransformed plants or there is achieved aninhibition of the synthesis of endogenous ammonium transporters wherebysuch cells show a reduced uptake of ammonium in comparison withuntransformed cells.
 14. Use of plant cells according to claim 13 forthe regeneration of whole plants.
 15. Use of the DNA sequences accordingto any one of claims 1-3, for the preparation of derivatives withchanged specificity of the ammonium transporter.
 16. Use of the DNAsequences according to any one of claims 1-3, for the isolation ofhomologous sequences from the genome of bacteria, fungi and transgenicplants.
 17. Use of the DNA sequences according to claims 1, 3 or 5, incombination with a transcription promoter in sense orientation, for theexpression of a translatable mRNA, which makes possible the synthesis ofan ammonium transporter in pro- and eukaryotic cells.
 18. Use of the DNAsequences according to claims 2, 3 or 5, in combination with atranscription promoter in anti-sense orientation, for the expression ofa non-translatable mRNA, which prevents the synthesis of an endogenousammonium transporter in pro- and eukaryotic cells.
 19. Use of the DNAsequences according to any one of claims 1-3 or 5 for the preparation oftransgenic plants with changed nitrogen metabolism.
 20. Use of the DNAsequences according to any one of claims 1, 3 or 5 for the preparationof transgenic crop plants, such as tobacco, potatoes, beets and maizewith increased yield.
 21. Use of the DNA sequences according to any oneof claims 2, 3 or 5 for the preparation of transgenic crop plants, suchas tobacco, potatoes, beets and maize under low input conditions. 22.Use of the DNA sequences according to any one of claims 2, 3 or 5 forthe preparation of transgenic crop plants, which also grow on acidsoils.